Project Summary Vibrio cholerae remains a major threat to world health in that cholera epidemics have ravaged Yemin, Africa, South Asia, and Haiti in only the last decade. This bacterial organism is also a powerful model system for understanding microbial pathogenesis and has ushered many new technologies for analyzing pathogen-host interactions. This proposal seeks a deeper understanding of how this mucosal pathogen interacts with commensal organisms, viral agents, and the host immune system. AI-018045 has previously supported V. cholerae research that has led to the discovery and characterization of cGAMP, a cyclic dinucleotide (CDN) and isomer of the product produced by cGAS (an immune sensor of clinical importance to autoimmunity and cancer). V. cholerae was used to engineer a form of the human cGAS enzyme that is locked in a DNA-bound, active conformation and this allowed its crystal structure to be solved facilitating anti-cGAS drug discovery. Previously, killing by the V. cholerae Type VI secretion system (T6SS) of commensal E. coli strains was found to induce an innate immune response that triggered early V. cholerae virulence gene expression in the host. The present proposal now offers creative experimental aproaches aimed at new challenges that have emerged from these discoveries including 1) understanding the biological activities of cGAMP made by the V. cholerae enzyme DncV including phage resistance and whether other novel cyclic and linear oligonucleotides synthesized by DncV-related enzymes discovered in the genomes of thousands of bacterial isolates have immunomodulatory activity and anti-viral function, 2) testing the hypothesis that cholera toxin (CT) production drives transmission through diarrhea or by reprogramming host metabolism so V. cholerae can take advantage of a CT-induced niche that is rich in hemin and long chain fatty acids, 3) understanding host innate immune activation that depends on the attack of commensal organisms by the T6SS of V. cholerae and 4) continuing the development of live attenuated probiotic-like vaccine HaitiV and Whole Cell Conjugate (WCC) vaccine technologies. State-of-the-art genetic, biochemical and cell biological approaches will be employed to address these topics which offer answers to questions such as i) what are the most important T6SS effectors and host pathways involved in T6SS commensal killing and induction of intestinal colonization by V. cholerae HaitiV? ii) Would commensal, pathogenic, or attenuated organisms loaded with with CDNs enhance T6SS-dependent host innate signaling and intestinal colonization of HaitiV? iii) Can a CRISPRi gene silencing technology developed for V. cholerae be used to enhance production of outer membrane vesicles which might deliver cGAMP or bacterial effector proteins into host cells? This proposal offers scientific impact that extends well beyond addressing cholera as a threat, and is likely to yield new discoveries that will reshape microbiological research in important areas of unmet medical need including antimicrobial vaccinology, immunomodulation, the host response to commensal microbiota, adjuvant research and drug development.